Multi-Omics Analysis Reveals Distinct and Combinatorial Roles of GABARAP-Family Proteins in Factor VIII Trafficking and Secreted Activity

The mammalian GABARAP subfamily (GABARAP, GABARAPL1, and GABARAPL2), a group of paralogs belonging to the ATG8 protein family, shares high sequence similarity, complicating assignment of paralog-specific functions. Coagulation factor VIII (FVIII), a large multi-domain glycoprotein and essential cofactor in the intrinsic coagulation cascade, places substantial energetic demands on the secretory pathway during biosynthesis. We investigated how the individual and combined loss of GABARAP-family members affects FVIII secretion and the cellular pathways that support this process, using previously established single-knockout FVIII-expressing HEK293 cell lines and newly generated double- and triple-knockouts (CRISPR-Cas9). Extracellular FVIII activity was assessed alongside intracellular co-localization with cellular markers, transcriptomic, proteomic, and bioenergetic analyses. Double knockouts displayed distinct combined effects on FVIII activity, whereas complete loss of the GABARAP family was associated with a qualitatively different response. Imaging revealed genotype-dependent alterations in FVIII localization across secretory, endosomal, and lysosomal compartments. Transcriptomic, proteomic, and multi-omics analyses identified genotype-associated signatures involving protein synthesis, stress responses, and energy metabolism. Cellular ATP content was positively associated with FVIII activity, while selected knockout clones exhibited altered respiratory and mitochondrial phenotypes. Together, these findings identify distinct, as well as interacting, associations between GABARAP-family paralogs and FVIII trafficking and secreted activity, with broader consequences for cellular metabolism.